rapid in the beginning of the experiment than when there has been an accumulation of the products of activity.

Such wide and varied abilities appertaining to an enzyme as are indicated by the foregoing summary demand that account be taken of its action, wherever natural changes of fat are in progress, and its ability to retain its activity for long periods of time and to work at low temperatures makes its rôle in fat changes in cold storage an important one.

Since enzymes of varied abilities have been traced to a bacterial origin it is quite reasonable to infer that those having some action on fats would be so produced. Schreiber," who has studied earth bacteria, was unable to find ferments, and considers the decomposition of fats to be dependent upon vital functions. It is then affected by all the conditions which affect life. Schreiber states that anaerobes split fats slightly, but their activity ceases at that point and is feeble at best. Rahn denies that anaerobes have any action on fats, but admits that they can decompose glycerin. Both of these authors report work on certain molds-Mucor, Penicilliumand find that they cause both a rapid and a complete decomposition. Among bacteria B. fluorescens liquefaciens shows a fat-decomposing power closely following that shown by molds. Rahn has determined this activity as expressed by the iodin number, which was considerably increased for palm fat, stearin, and butter fat. Laxa d has recorded analyses covering the values usually determined in fat examinations using butter fat with molds, yeasts, and fat splitting bacteria proper. The latter, with the exception of B. fluorescens liquefaciens, commonly cause a decrease in the iodin number; the other fungi noted usually increased it. The Reichert-Meissl number was but little changed. The acid value increased for both molds and bacteria. The saponification number decreased with both. The index of refraction in many instances fell when the iodin number rose, and when the latter decreased the refractive index remained stationary. The Hehner number for molds showed a rise.

It will be observed that there is a distinct difference between the action of molds and of bacteria on fats. This is again expressed in the work of Girard, who believes that Aspergillus and Penicillium act by way of an enzyme and Laxa has demonstrated by experiment that such is the case. With substances as complex as are fats in their natural environment and with forces as delicate as fungi

a Fettzersetzung durch Microorganismen, Arch. Hyg., 1902, 41: 328. Die Zersetzung der Fette, Centrbl. Bakt., 2 Abt., 15: 53.

c Loc. cit., 15: 422.

dUeber die Spaltung des Butterfettes durch Microorganismen, Arch. Hyg., 1902, 41: 118.

e Loc. cit.

49078-Bull. 115-08-7

and enzymes, and with such universal factors as air and light to be considered, it is not to be wondered at that the whole question of fat decomposition in nature is still greatly in need of elucidation. The literature on the subject, as indicated in the foregoing discussion, is chiefly concerned with plant oils and butter fat and with fats freed from the tissues normally occurring with them. Almost invariably, also, the studies made have been conducted at body or at room temperature. The discussion of the changes in the fat of coldstored chickens as indicated by the variation between their values and those of the fresh birds, which values are given in another section of this report, becomes therefore a difficult task. Light, as a cause of decomposition, need scarcely be considered; air, at least for the superficial fats, is probably a factor and would tend to increase the acidity. So great are the differences between the coldstored and fresh chickens when the acid value, saponification number, and ester number are considered that probably not only air but enzymes as well, and perhaps bacteria too, have all played a part in the alteration. The lowering of the iodin number rather argues for a certain amount of bacterial action, as does also the decrease of the saponification number, since the available contributions on the subject agree in assigning such results to bacteria. The change in the Hehner number due to bacterial action is not discussed to any extent in the literature. Laxa," in his study of butter, makes two determinations, one for Oidium lactis and the other for B. fluorescens liquefaciens, finding an increase in both cases. The Hehner number for fresh chicken fat is lower than is usually found for animal fats, and after cold storage a further decrease is observed. It is difficult to explain the cause of a rise in the Hehner number as a result of fungoid action, since that is essentially katabolic, and not at all of the character to produce acids of a higher type than those normally present, whereas the decomposition of insoluble acids with the production of soluble forms is perfectly logical and quite in line with the observations of Rahn and Krueger.

SUMMARY OF RESULTS.

(1) Macroscopic observations of fresh and cold-stored chickens show that certain plainly visible differences exist between the two classes, which differences are progressive, depending on the length of the storage period.

(2) Chemical analyses of fresh and cold-stored chickens, wherein were determined the total nitrogen for both dark and light meat and its distribution between the classes of compounds commonly accepted as the result of protein hydrolysis, the various values from which a knowledge of the composition of fat may be obtained and such

a Loc. cit.

minor constituents as water, ash, etc., have served to show that for the protein distribution there is a slight variation in the cold-stored product from the fresh, and for the fat values a wide variation.

(3) A histological examination of the muscle of both fresh and cold-stored chickens shows a marked and a progressive change in the structure of the fibers which is deep-seated and after long periods renders the tissue almost unrecognizable. Selective microchemical differentiation of the tissues confirms the chemical change found by gross analytical methods.

(4) A bacteriological examination of fresh and cold-stored chickens reveals the presence of appreciable numbers of organisms, calculated on the gram basis, in the edible portions of those preserved by cold, though the numbers were not large. In fresh fowls the same technique gave no bacterial growth.

GENERAL REVIEW OF THE INVESTIGATION.

In reviewing the details of this preliminary work it must be remembered that some of the inferences drawn may be modified by future investigations, although the fact that certain changes take place in the food materials examined after definite periods of storage is well established both by the organoleptic and by the chemico-bacteriological tests and is further confirmed, in the case of the chickens examined, by the histological studies.

The principal lines of investigation reported, namely, on eggs, quail, and chickens, conducted at Washington under known conditions. of storage, and the more complete study of chickens stored under. market conditions at Philadelphia, are summarized in the following pages, brief reference also being made to the conclusions drawn from the milk investigation previously reported by Pennington.

ORGANOLEPTIC TESTS ON CHICKENS AND QUAIL.

The general results of the organoleptic tests of the stored fowls and birds leads to the conclusion, first, that even after three months, before cooking, it is not difficult to distinguish, by the appearance, color, and odor, a freshly killed bird from the one that has been in cold storage. The shriveled condition of the eye and the skin and the generally dilapidated appearance of the bird are very significant and distinct. After cooking, however, within a period of three months, there is much disagreement on the part of the jurors respecting the proper classification thereof. This is especially true as to distinguishing between the drawn and the undrawn birds, where the variations in judgment are of such a character as to lead to the belief that it is impossible within that time, from the taste, odor, and smell of the cooked bird, to determine which one had been drawn and which was undrawn at the time of storage. The possibilities of determining

which is the fresh bird are very much greater, even after a lapse of three months, although occasional mistakes may be made in this respect also. At later dates, such as at the end of six months, nine months, or twelve months, the difference between the birds becomes more and more pronounced, so that it may with certainty be said that even after cooking one would rarely confound a fresh bird with one which had been in storage six months. At that date the flavor and the general character of the meat have so deteriorated that it is not difficult to distinguish between the fresh and the stored fowl. Even at this date, however, there is some difficulty in distinguishing between the drawn and the undrawn bird. At the end of a year or more it would be quite impossible to make a mistake in most cases between the fresh and the stored birds.

Summing up the organoleptic properties, it may be said that for a short time, possibly six weeks or even longer, there is no perceptible change produced in a chicken by having it frozen. There certainly does not seem to be any evidence that it is better, and there is no convincing evidence that it is any worse. After three months, however, the fresh chicken is easily distinguished by its properties, as a rule, from the cold storage chicken, even after cooking, and to an absolute certainty before cooking. This distinction between the fresh and the stored bird becomes more and more marked as the time of storage is increased. In so far as the drawn and undrawn chickens are concerned there is much less certainty of being able to distinguish between them. However, 70 per cent of the jurors were able to pick out the undrawn bird by its stronger odor and taste after a storage period of from six to fifteen months, but at the test representing 18.5 months' storage the two birds were about equally dry and tasteless.

The general conclusion is, therefore, that in the case of frozen birds there is no indication of any improvement in quality, that is, in taste, odor, or flavor, during cold storage. There is a deterioration which is noticeable, even at the end of three months, and becomes more marked as the time of storage grows longer. Hence, without any reference whatever to the question of wholesomeness, cold storage prolonged for six months or more appears to be distinctly detrimental as far as taste, flavor, and palatability are concerned.

BACTERIOLOGICAL AND CHEMICAL INVESTIGATIONS.

MILK.

As a part of this investigation certain studies have been made on the changes taking place in milk when kept at low temperatures. The detailed report of this study has already been published," hence

a J. Biol. Chem., 1908, 4: 353, Bacterial Growth and Chemical Changes in Milk Kept at Low Temperatures, M. E. Pennington.

1

there will be included here only the conclusions reached, which are as follows:

SUMMARY OF RESULTS.

Bacteria in milk increase in numbers when the temperature is maintained at or a little below 0° C. This temperature is below that ordinarily assigned as the limit of bacterial multiplication.

Milk has been kept for periods ranging from a few days to almost two years at a temperature of 29° to 31° F., and also at 32° F. It has been kept in a packages of ten quarts and one quart. It has been the cleanest milk obtainable, by the most carefully enforced refinements of modern dairying; and it has also been market milk produced in the ordinary dirty stable and subjected in transit to the usual careless handling.

Bacterial growth at the end of a week, even in the cleanest milk, which contained as low as 300 organisms to the cubic centimeter, was pronounced. There was a steady increase in the number of organisms for five or six weeks, and at their maximum they numbered hundreds of millions. Occasionally they passed the billion mark per cubic centimeter.

Continued exposure to a temperature of 29° to 31° F. causes, after a lapse of from seven to twenty-one days, the formation of small ice crystals which gradually increase until the milk is filled with them and there may be an adherent layer on the walls of the vessels. The milk does not freeze solidly. In spite of the fact that the milk was a semisolid mass of ice crystals, the enormous increase in bacteria which this study shows took place. Though the bacterial content was numerically in the hundreds of millions per cubic centimeter there was neither odor nor taste to indicate that such was the case. Neither did the milk curd even on heating, and it was not until the bacterial content began to fall, and organisms of putrefaction were under way, that the use of the milk for household purposes would, to the ordinary observer, become contra-indicated.

A classification on a chemical basis of the organisms occurring at these low temperatures shows that there were constantly present bacteria which formed acid and bacteria which acted upon proteid. There were also neutral organisms, which formed neither acid nor alkali and did not act upon gelatin. The acid-forming organisms were generally in relatively smaller numbers than are found when milk is kept at higher temperatures, and the liquefying organisms were more numerous. Certain species, such as B. formosus, B. solitarius, and B. raveneli, were especially resistant to cold and frequently were the predominating species, or almost in pure culture at the close of the experiment.

A very marked difference in both the number and kind of organisms which developed on the plates was noticed, depending upon the temperature at which the plate was incubated. In certain experiments the maximum number grew at 37° C. In others the temperature at which the milk was stored served best for colony formation. The relative number of organisms growing at 37° C., 20° C., and 0° C., or a little below, varied greatly also with the length of time that the milk had been kept in storage, the organisms developing at body temperature being ordinarily greatly in excess at the beginning of the experiment and diminishing until near its close, when a sharp rise was apt to take place.

The determination of the acidity showed that after a few weeks a much higher acid content was reached than is ordinarily required for the spontaneous separation of curd, which, however, seldom happened. Milk having this high acidity, when placed in an ordinary ice chest, increased in acid content but did not curd for days after exposure to the higher temperature.

The chemical study of the proteid of milk in cold storage showed that the casein was rapidly digested until finally more than 50 per cent of it was changed to soluble compounds. Caseoses, amido acids, and probably peptones, increase apparently at